Isolation of viruses, including mollivirus, with the potential to infect Acanthamoeba from a Japanese warm temperate zone

Acanthamoeba castellanii is infected with diverse nucleocytoplasmic large DNA viruses. Here, we report the co-isolation of 12 viral strains from marine sediments in Uranouchi Inlet, Kochi, Japan. Based on the morphological features revealed by electron microscopy, these isolates were classified into four viral groups including Megamimiviridae, Molliviridae, Pandoraviridae, and Pithoviridae. Genomic analyses indicated that these isolates showed high similarities to the known viral genomes with which they are taxonomically clustered, and their phylogenetic relationships were also supported by core gene similarities. It is noteworthy that Molliviridae was isolated from the marine sediments in the Japanese warm temperate zone because other strains have only been found in the subarctic region. Furthermore, this strain has 19 and 4 strain-specific genes found in Mollivirus sibericum and Mollivirus kamchatka, respectively. This study extends our knowledge about the habitat and genomic diversity of Molliviridae.


Introduction
Acanthamoeba are free-living protists that are widely distributed in the environment.These protists incorporate bacterial cells into phagosomes via pseudopod extension.In this step, certain viruses are also incorporated into Acanthamoeba cells, and cell lysis occurs in some cases [1,2].Owing to this unique feature, several Acanthamoeba species, including A. polyphaga and A. castellanii have been used as hosts to isolate viruses.
NCLDVs are known to have a set of highly conserved genes (core genes).These core genes encode proteins involved in important cellular processes such as nucleotide synthesis, DNA replication, DNA recombination and repair, and transcription [13].Phylogenetic analysis based on the presence or absence of these genes suggested that NCLDVs are monophyletic and represent a fourth domain of life that originated from a common ancestor [14].Furthermore, recent metagenomic analysis predicted that horizontal gene transfer occurs between various NCLDVs and host eukaryotes [15].Therefore, NCLDVs are important biological entities for understanding evolutionary processes and ecological networks [2,15].
These NCLDVs universally and heterogeneously exist in marine environments [16].For example, high proportions of unique NCLDVs are present in the polar biomes [16].Likewise, Molliviridae were only isolated from the subarctic region in areas such as Siberia and Kamchatka [10,17].Therefore, investigating NCLDVs in different locations is essential for elucidating the marine ecosystem dynamics.
Uranouchi Inlet is a small semi-enclosed sea area located at the southeastern side of Shikoku Island, Japan.Although the existence of diverse Mimiviridae in the inlet was revealed [18], the Acanthamoeba viruses have not been isolated to date.Here, we isolated and characterized Acanthamoeba viruses of four families from Uranouchi Inlet, Japan.

Isolation of lytic agents causing A. castellanii cell death
Soil samples were collected in Uranouchi Inlet, Kochi Prefecture, Japan from 22 August 2019 through 30 July 2020 (S1 Fig) .Permission for sampling for this study was obtained from the Japan Coast Guard and Kochi Prefecture.Up to 3 g of the sample was suspended in a 10-fold volume of distilled water, and then stirred at room temperature for 1 h.The suspended samples were incubated at 4˚C until they naturally settled.Each supernatant was filtered through 5.0-μm pore size cellulose membrane (150 mm, ADVANTEC, Tokyo, Japan).An aliquot (50 μL) of filtrates was inoculated into A. castellanii cultures (250 μL) in 96-well plates (Thermo Fisher Scientific, MA, USA) and incubated for a week under the above conditions.
Each well was monitored by optical microcopy every other day.The cell lysates were diluted with distilled water; then, serial 10-fold dilutions (250 μL) were inoculated into A. castellanii (150 μL) cultures in 96-well plates.After incubation for a week, this extinction dilution procedure was conducted again with the lysates from the most diluted well.

Verification of bacterial absence from the lysate
Five media were prepared for sterility tests using PYG medium following instructions from the National Institute for Environmental Studies (B-I, B-II, B-IV, B-V, and YT; https://mcc.nies.go.jp/02medium.html).The lysates (10 μL) were inoculated into each medium (500 μL) and incubated in 48-well plates (Thermo Fisher Scientific) at 26˚C.Each well was monitored by optical microscopy every other day.Additionally, the lysates (100 μL) were filtered through 0.2-μm (ADVANTEC) and 0.1-μm (Pall Corporation, NY, USA) syringe membrane filters, respectively, and then inoculated into A. castellanii culture (150 μL) to confirm whether the filtrates retained the lytic activity.
DNA was extracted from the lysates using the phenol/chloroform/isoamyl alcohol procedure after incubation with 10% (v/v) SDS (Tokyo Chemical Industry Co., Ltd., Tokyo, Japan) and protease K (FUJIFILM Wako) for 1 h at 56˚C.The extracted DNA was subjected to PCR amplification of the 16S rRNA gene using Ex Taq (TaKaRa Bio Inc., Shiga, Japan) with 27F and 1492R primers [19].PCR conditions were as follows: initial denaturation at 94˚C for 2 min, followed by 35 cycles of 94˚C for 30 s, 55˚C for 30 s, and 72˚C for 30 s; and a final extension at 72˚C for 7 min.The resultant products were confirmed by electrophoresis on a 2% agarose gel.

Electron microscopy
After incubation for two weeks, the lysates were prepared for electron microscopy.An aliquot (35 μL) of the samples was mixed with 5 μL of 4% osmium tetroxide, and then incubated for 5 min at room temperature.The fixed samples were collected on a 0.2-μm membrane filter (25 mm), washed twice, and immersed in distilled water at 4˚C overnight.The filters were immersed in 30%, 50%, 70%, 90%, and 95% ethanol every 5 min.Then, the filters were immersed in 100% ethanol three times for 20 min at room temperature and completely dried using a critical point dryer (JEOL JCPD-5).The dried samples were coated with osmium tetroxide by an osmium coater (Neoc-Pro, Meiwafosis Co., Ltd., Tokyo, Japan) and observed using field-emission scanning electron microscopy (FE-SEM; JEOL JSM-6500F).
After culturing for one week, A. castellanii (150 mL) was mixed with 20 mL of each lysate, and then incubated for 24 h under the above conditions.An aliquot (42.5 mL) of the cultures was centrifuged at 600 g for 10 min at 4˚C.The cell pellet was washed with 0.1 M phosphate buffer (pH6.8) and suspended in the same buffer (460 μL).The samples were fixed in 25% glutaraldehyde (Nacalai Tesque) at a final concentration of 2% and incubated for 1 h at 4˚C.After washing twice, the fixed samples were resuspended in 500 μL of phosphate buffer and stored at 4˚C until analysis.After centrifugation at 1,100 g for 5 min, the samples were mixed with 1 mL of distilled water containing 1% (w/v) Agarose-S (Nippon Gene Co., Ltd., Toyama, Japan) and solidified during centrifugation again under the same conditions.The agarose blocks with A. castellanii cells were cut into 1-mm cubes and then immersed in 500 μL of phosphate buffer.
Ultrathin sections of each sample were prepared by Dr. Kenichi Yagyu as follows.After washing with the buffer, the samples were fixed with 0.1 M phosphate buffer (pH7.3)including 1% osmium tetroxide at 4˚C for 1 h, followed by dehydration treatment with ethanol.The treated samples were coated with Epon 812 (TAAB Laboratories Equipment, Reading, UK), and then ultrathin sectioned using a Leica EM UC7 microtome (Leica Microsystems, Wetzlar, Germany).After double staining with uranium and lead dye, the samples were observed using transmission electron microscopy (TEM; JEOL JEM-1400Plus).

Viral genome sequencing, assembly, and phylogenetic analyses
After culturing for one week, A. castellanii (80 mL) were infected with each viral lysate (800 μL), and then incubated for two weeks.After centrifugation at 200 g for 5 min, the supernatants were further centrifuged at 20,400 g for 20 min.The samples were resuspended in 900 μL of distilled water, and then subjected to DNA extraction using the above-mentioned method.DNA libraries were prepared using the NEBNext Ultra II FS DNA Library Prep Kit for Illumina (New England Biolabs, MA, USA) according to the manufacturer's instructions.Library sequencing (2 × 150-bp read length; NovaSeq 6000) was performed by Rhelixa Co., Ltd.(Tokyo, Japan).After adapter trimming and quality filtering (Q30), total reads from each sample were assembled using SPAdes version 3.15.3 with default k-mer lengths [20].Detection of the viral signal was performed using VirSorter 2.2.3 with the "-include-groups NCLDV" option [21].ViPTree server version 3.4 was used for proteomic tree construction, gene annotation, and genomic alignment views [22].The genomic similarity score (S G ) value was set to � 0.15 (viral genus level cut-off) according to a previous study [23].Maximum likelihood analysis of the core genes coding DNA polymerase family B and VVA18 helicase was performed using the Molecular Evolutionary Genetics Analysis (MEGA) package version 11.0.13[24].

Isolation of lytic agents from soil samples
During the survey period, we collected 31 soil samples from sediments at multiple stations in Uranouchi Inlet (S1 Fig).Among these samples, nine inocula showed lytic activity against A. castellanii (S1 Table ).After purification by the extinction dilution method, we isolated 12 lytic agents that caused A. castellanii cell death (S1 Table ).
Sterility tests of lytic agents showed no increase in turbidity in the inoculated five media due to the propagation of microbial cells.Likewise, agarose gel electrophoresis did not indicate a band corresponding to the 16S rRNA gene.Furthermore, the filtered agents showed no lytic activities against A. castellanii cells.Based on these results, we concluded that lytic activities could originate from giant viruses but not microorganisms or bacterial viruses.2D-2F), which was consistent with morphological features of known molliviruses [7].The two isolates Me1-1 and Me1-2 also showed unique morphological features of Megamimiviridae, including a large capsid (avg.0.45 μm in diameter) with fibrous structures (Figs 1I, 1J and 2G-2I) [25].The other isolates, Ce2-1 and Ce7-1, had morphological features consistent with cedratviruses, including ovoid particles (avg.major and minor axes: 1.2 and 0.7 μm, respectively) with a cork-like structure at both ends (Figs 1K, 1L and 2J-2L) [9].

Genome analysis of A. castellanii viruses
To reveal the genomic features, we next sequenced and assembled 12 genomes of A. castellanii viruses (� 10 kb) isolated from Uranouchi Inlet.Acanthamoeba castellanii viruses were largely classified into four groups using a viral proteomic tree [26] based on their genome similarity scores derived from tBLASTx scores (Fig 3) [22].

Discussion
In this study, we isolated the giant viruses infecting A. castellanii from marine sediments.Our results indicated that four phylogenetically distinct viral groups (Megamimiviridae, Molliviridae, Pandoraviridae, and Pithoviridae) coexist in Uranouchi Inlet, Kochi, Japan.In particular, sampling station 1, which is located at the closed-off section of the inlet, was a "hot spot"  ).However, we could not isolate giant viruses closely related to Asfarviridae [4], Marseilleviridae [5], and Medusaviridae [6].
All giant viruses isolated in this study showed high similarities to known viral genomes taxonomically clustered together (Figs 3-5).However, it is worth noting that mollivirus Mo1-1 was isolated from the sediment in Uranouchi Inlet.To date, Molliviridae has only been isolated from the subarctic region, such as from the permafrost layer and Russian riverbank [7,17].Therefore, this is the first report on the existence of Molliviridae in a warm temperate zone and endorses that they are not extinct from the current environment [17].
In the M. kamchatka genome, 96% of the encoded proteins were highly conserved compared with those of M. sibericum which was in a dormant state for 30,000 years; this indicates that most of these proteins contribute to viral fitness [17].The highly conserved genome of Mo1-1 also supported the importance of these proteins for the mollivirus lifecycle (Fig 5A).Meanwhile, the Mo1-1 genome contains not only unique genes found in the M. sibericum genome, but also M. kamchatka-specific genes [17] (S3 Table ).Further studies are needed to elucidate acquisition/loss events of these genes and their contribution to viral fitness for each Molliviridae strains.
In conclusion, we revealed the diversity and genomic features of A. castellanii viruses in Uranouchi Inlet, Japan.These findings expand our current knowledge regarding Molliviridae habitat and genomic differences among the strains.The results of this study will provide an opportunity to better understand the evolution and diversity of Molliviridae if they are isolated from a wide range of climatic zones in the future.

Fig 3 .
Fig 3. Proteomic tree of 12 A. castellanii viral genomes isolated in this study and 533 related eukaryotic dsDNA viruses.(A) Whole proteomic tree, including 534 related eukaryotic dsDNA viruses, generated by ViPTree server version 3.5.The dendrogram represents the proteome-wide similarity relationships among the 12 A. castellanii viruses isolated in this study (red branches) and reference viral genomes (black branches).Branch lengths are shown on a logarithmic scale from the root of the entire tree.(B) Inner and outer rings that are outside the dendrogram represent viral family classifications and taxonomic groups of known hosts, respectively.(C) Enlarged view of the proteomic tree that includes the viruses isolated in this study.https://doi.org/10.1371/journal.pone.0301185.g003